Fluorescence is widely used in analytical and clinical chemistry (Schulman, 1993; Spichiger-Keller, 1998; Wolfbeis, 1991a; Wolfbeis, 1991b; Kunz. 1996; Lakowicz, 1994a; Thompson, 1997). In the past several years there has been increasing interest in the use of time-resolved fluorescence as an analytical tool, e.g., for non-invasive sensing (Szmacinski and Lakowicz, 1994a; Lippitsch et al., 1997; Lippitsch et al., 1988; Bambot et al., 1995; Spichiger-Keller, 1998; Fraser, 1997). The basic idea is to identify fluorophores or sensing schemes in which the decay time of the sample changes in response to the analyte, and to use the decay time to determine the analyte concentration. Such lifetime-based sensing is most often performed using the phase-modulation method. The use of phase angles or decay times rather than intensities is advantageous because decay times are mostly independent of the signal level and can be measured in turbid media and even through skin (Bambot et al., 1995; Szmacinski and Lakowicz, 1996).
Most fluorophores used for sensing display decay times on the nanosecond timescale. It now appears possible to design low cost instruments for sensors with ns decay times. For instance, it is known that blue and UV light emitting diodes (LEDs) can be modulated to over 100 MHz and used as the excitation source in phase-modulation fluorometry (Sipior et al., 1997; Sipior et al., 1996; Lakowicz et al., 1994a; Randers-Eichorn et al., 1997). However, it may be useful to avoid the use of frequencies near 100 MHz which are needed for ns decay time measurements, and thus use the simpler electronics for lower frequencies. Also, a significant fraction of sensing fluorophores display changes in intensity without changes in lifetime.
What are the advantages of low frequency modulation sensing? It is now accepted that lifetime-based sensing can be preferable to intensity-based sensing because the lifetimes are mostly independent of changes in probe concentration and/or signal level. Modulation sensing shows many of these advantages. The modulation will be independent of the total signal level. Hence, modulation sensing can be accurate even if the overall signal level changes due to flexing in fiber optics or changes in the positioning of the sample. However, it is necessary that the relative proportions of the short and long lifetime fluorophores remain the same. If the relative intensities change, in a manner independent of analyte concentration, then the modulation calibration curve will also change. Hence, the calibration curves for a modulation sensor will change if the sensing and reference fluorophore photobleach at different rates.
An advantage of low frequency modulation sensing is the simple instrumentation One can imagine simple hand-held instruments for modulation intensity measurements (FIG. 1). It is now well known that light emitting diodes can be easily modulated at frequencies up to 100 MHz (Sipior et al., 1997; Sipior et al., 1996; Lakowicz et al., 1994a; Randers-Eichorn et al., 1997). Also, LEDs are available within a range of output wavelengths, even down to the near UV at 390 nm (Sipior et al., 1997). LEDs consume little power and can easily be driven by batteries. Hence, the modulation sensor could be a small device held near the skin. The long-lifetime complex can be part of the device, so none of the long-lifetime probe enters the sample or tissue. The high chemical and photochemical stability of the metal-ligand complexes suggests the signal from the long lifetime reference will be constant for long periods of time. Hence, such devices may prove valuable for quantitation of intrinsic and extrinsic fluorophores in tissues.
Additionally, there has been considerable progress in the design and synthesis of long lifetime metal-ligand complexes. Ruthenium, osmium and rhenium complexes have been reported (Lakowicz et al., 1995; Terpetschnig et al., 1997; Castellano et al., 1998). The rhenium complexes are particularly useful in that they display high quantum yields and lifetimes up to 3 xcexcs in oxygenated solution (Guo et al., 1998; Guo et al., 1997).
And finally, the most important advantage of modulation sensing may be the expanded range of analytes. Any sensing fluorophore which changes intensity can be used in this model. A change in probe lifetime is not needed. Hence, modulation sensing can be used with probes such as a sodium-binding benzofuran isophthalate (SBFI) and a potassium-binding benzofuran isophthalate (PBFI), which are poor wavelength-ratiometric probes for sodium and potassium.
The possibility of non-invasive sensing is based on the low absorbance of tissue at red and near-infrared (NIR) wavelengths, and the increasing availability of long wavelength fluorophores (Lakowicz, 1994b; Matsuoka, 1990; Leznoff and Lever, 1989). However, it is difficult to perform intensity measurements in highly scattering media (Oelkrug, 1994), which has led to interest in the use of time-resolved fluorescence and lifetime-based sensing for non-invasive fluorometry (Szmacinski and Lakowicz, 1994a; Hutchinson et al., 1995; Szmacinski and Lakowicz, 1994b). It is now known that fluorescence lifetimes can be measured in the presence of extensive light scattering, and can even be measured through skin (Bambot et al., 1995).
The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice, are incorporated by reference, and for convenience are respectively grouped in the appended List of References.
Described here is a new method which allows quantitative measurements of fluorescence intensity with simple instrumentation. The method is self-calibrating related to a reference fluorophore. The method can also be used in highly scattering media. The measurement principle is based on observing the emission from both the fluorophore of interest with a ns decay time and of a reference fluorophore which displays a much longer microsecond lifetime. The reference fluorophore is placed on rather than in the sample to mimic a sensing device with the long lifetime reference held against the skin. The amplitude modulation of the emission is observed using the standard method of frequency-domain fluorometry. At an intermediate modulation frequency, the modulation is equivalent to the fractional intensity of the ns fluorophore. The method was tested in 0.5% intralipid, which is more highly scattering than skin. Quantitative intensity measurements were obtained for various concentrations of fluorescein in intralipid, and of the pH sensor 6carboxy fluorescein. Low frequency modulation measurements provide a general method for quantitative measurements in the presence of factors which preclude direct intensity measurements, or applications which require simple internally referenced measurements.